Investigating the cytoadherence of plasmodium falciparum infected red blood cells

INVESTIGATING THE CYTOADHERENCE OF PLASMODIUM FALCIPARUM INFECTED RED BLOOD CELLS ZHANG ROU (B.Eng. (Hons), NTU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ADVANCED MATERIALS FOR MICRO- AND NANO- SYSTEMS (AMM&NS) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements During the four years as a Ph.D student working in the Singapore-MIT alliance, I owe a great many thanks to many people. First, I would like to express my deepest thanks to my supervisor, Prof. Lim Chwee Teck, for his valuable guidance throughout my Ph.D project and thesis. I would also like to thank to my former co-supervisor, Prof. Subra Suresh, and my thesis co-supervisor, Prof. Cao Jianshu and Dr. Dao Ming, for their helpful discussions and suggestions in helping me finish my projects. I would also like to express my deepest thanks to all my colleagues and friends in Nano Biomechanicss Lab (NUS), Suresh Group (MIT) and the Infectious Disease Group (SMART) for their valuable suggestions and help. Personally, I would like to express my deepest gratitude to Dr Monica Diez-Silva for her continuous help and suggestions in my research. I would also like to thank the Singapore-MIT Alliance for their financial support for my graduate studies. i Table of Contents ACKNOWLEDGEMENTS . I TABLE OF CONTENTS II SUMMARY .IV LIST OF TABLES .VI LIST OF FIGURES . VII LIST OF ABBREVIATIONS . XIII LIST OF SYMBOLS . XV CHAPTER 1. INTRODUCTION 1.1 Malaria . 1.2 Pathogenesis of Plasmodium falciparum . 1.3 Current Studies on the Mechanical Properties and Cytoadherence of Infected RBCs 1.3.1 Mechanical Properties of Malarial-Infected RBCs . 1.3.2 Cytoadherent Properties of Infected RBCs 14 1.4 Hypothesis, Objectives and Scope of this Thesis 25 1.4.1 Hypothesis . 25 1.4.2 Objectives 27 1.4.3 Scope of Work . 28 CHAPTER 2. CYTOADHERENCE STUDY OF PLASMODIUM 30 FALCIPARUM-INFECTED RBCS TO CSA 30 2.1 Experimental Methods and Materials 31 2.1.1 Sample Preparation . 31 2.1.2 Adhesion Force and Energy Density Measurements 36 2.1.3 Cell Deformability Measurements . 46 2.1.4 Statistical Analysis 48 2.2 Results 48 ii 2.2.1 Examination of Binding Receptor on Testing Cells . 48 2.2.2 Determination of Cell-Cell Contact Duration . 50 2.2.3 Adhesion Measurement Tests at Different Asexual Stages 52 2.3 Discussion 70 CHAPTER 3. EFFECTS OF FEBRILE TEMPERATURE ON CYTOADHERENCE 77 3.1 Experimental Methods and Materials 79 3.1.1 Sample Preparation . 79 3.1.2 Temperature Controlling and Cell Viability Assays . 80 3.1.3 Adhesion Force and Energy Density Measurements 82 3.1.4 Imaging the Expression of Binding Ligands . 82 3.1.5 Cell Deformability Measurements . 83 3.1.6 Statistical Analysis 83 3.2 Results 83 3.2.1 Effects of Febrile Temperature on Cell Viability 83 3.2.2 Adhesion Measurements after Febrile Temperature Incubation . 87 3.2.3 Parametric Studies 95 3.3 Discussion 110 CHAPTER 4. CONCLUSIONS AND FUTURE STUDIES . 114 4.1 Conclusions 114 4.2 Future Studies 117 REFERENCES 120 iii Summary Malaria infects 300-500 million people and results in 1-3 million deaths each year. During malaria infection, the malaria parasites invade the human red blood cells (RBCs) and in the process, modify the mechanical and adherent properties. The deformable RBCs can become stiff and sticky, and bind to the endothelial cells which results in vascular occlusion. The cytoadherence of malaria-infected red blood cells (iRBCs) is directly related to the malaria severity, and the occlusion leads to several symptoms, such as pain, organ damage, or even death. In this thesis, the cytoadherence of iRBCs in terms of the adhesion force, adhesion percentage and energy density, were studied between the FCR3-CSA iRBCs and CSA-expressing Chinese hamster ovary (CHO) cells using the dual micropipette step-pressure technique. The adhesion force was systematically studied (1) among different asexual stages from ring to schizont stages and, (2) under the effect of febrile temperature. In studying the adhesion force at different asexual stages, a significant increase in both the adhesion force and percentage of adhesion was observed from the early trophozoite to early schizont stage. However, at late schizont stage, both the adhesion force and the percentage of adhesion decreased significantly. In studying the effect of the febrile temperature on iRBC cytoadherence, it was found that h incubation at febrile temperature could significantly increase both the adhesion force and percentage of adhesion. iv However, a longer incubation at febrile temperature leads to significant cell death. The adhesive ligand density and cell rigidity were proposed to be factors affecting the adhesion contact area, which was proportional to the resultant adhesion force. The microscope images were used to examine the adhesion contact area. The mean fluorescent intensity (MFI) obtained from the flow cytometric analysis was used to quantify the surface ligand density. Moreover, the cell membrane shear elastic modulus was measured using the micropipette aspiration technique. It was found that while the resultant adhesion force was proportional to the surface ligand density, and inversely proportional to the cell rigidity, the ligand density played a major role in affecting the resultant adhesion force. Less than 30% change in the shear elastic modulus could not significantly change the adhesion force, and when more than 100% change in shear elastic modulus occurred from the early schizont to late schizont stage, the resultant adhesion force decreased significantly. The results of this study could potentially provide valuable information in better understanding the cell-cell adhesion and the factors involved in affecting the resultant cell-cell adhesion force during the pathophysiology of this disease. v List of Tables Table Table 2.1. Table 2.2. Table 3.1. Table 3.2. Page Voltmeter reading adapted from one experiment data where voltage readings were obtained at different water levels or volume markers on the Falcon pipet. 42 Summary of the adhesion percentage, force, contact diameter and the adhesion energy density measured between the iRBCs and CHO cells at room temperature. 68 Summary of the adhesion percentage, force, contact diameter and the adhesion energy density measured between the iRBCs and CHO cells at different temperatures. 108 Summary of the adhesion percentage, force, contact diameter and the adhesion energy density measure between the iRBCs and CHO cells after the PfEMP1 and/or PS was blocked. 109 vi List of Figures Figure Page Figure 1.1. Global distribution of malaria (WHO 2004). Figure 1.2. Life cycle of malaria parasites, from infection, asexual cycle, sexual cycle to transmission back to mosquito (Miller et al. 2002). The biconcave shape of normal red blood cells observed under the optical microscope (scale bar = µm). Schematic drawing of nRBCs membrane structure, including lipid bilayer, band transmembrane proteins, spectrin filaments and junction complex proteins (Maier et al. 2009). Schematic drawings of (A) healthy RBC spectrin network proteins and (B) knob structure of malaria-infected RBC (Maier et al. 2009). 13 Structures and sub-domains of PfEMP1 variant proteins, and sub-domains responsible to bind to different host receptors such as heparin sulfate, CR1, CD36, ICAM-1, CD31, CSA, IgM etc (Kraemer et al. 2006). 17 Schematic representation of PfEMP1 induced cytoadherence lead to several malaria symptoms, such as severe malaria, cerebral malaria, placental malaria etc (Kraemer et al. 2006). 18 Borosilicate glass tubings with tips forged into 1-2 μm micropipettes. 36 Sample holding chambers made from two cover glass and parafilm separators. 37 Schematic drawing of micropipette system for dual pipette technique (two micropipettes). The cell mounting chamber with the sample was mounted on a microscope for observation, and the micropipettes were connected to a pressure controlling water column system. Pressure was read by a pressure transducer, and the height of water column was controlled by an externally connected syringe pump. 38 Figure 1.3. Figure 1.4. Figure 1.5. Figure 1.6. Figure 1.7. Figure 2.1. Figure 2.2. Figure 2.3. Figure 2.4. Optical microscope images detailing the dual pipettes steppressure technique. (1) One iRBC was placed on the CHO cells for a contact duration. (2)-(12) The aspirating pressure in the left micropipette was increased (P1 < P2 < P3 < P4 < vii Figure 2.5. Figure 2.6. Figure 2.7. Figure 2.8. Figure 2.9. Figure 2.10. Figure 2.11. Figure 2.12. P5 < P6) and attempts were made to pull the iRBC away from the CHO cell. (13) When the aspirating pressure or force applied was higher than the adhesion force, the iRBC was then able to detach from the CHO cell. 40 Schematic drawing of determining the zero pressure. When the RBC is neither aspirated into the micropipette nor blown away, the aspiration pressure is the same as the atmospherical pressure. It is recorded as at zero aspiration pressure. 41 Schematic representation of adhesion energy density calculation of two adhering cells. 44 (A) Schematic drawing of a cell being aspirated. The micropipette inner diameter RP, the length of elongation , and the aspiration pressure are used to calculate the membrane shear elastic modulus. (B) Optical microscope image of a human RBC being aspirated (scale bar = μm). 45 Plot of aspirated length vs suction pressure from one of the micropipette aspiration experiments. A linear relationship between aspirating pressure and aspirated length is shown. 47 Optical and fluorescence microscopic images of Alexa Fluor 555 stained PfEMP1 on the mature stage iRBCs. (A). PfEMP1 proteins were evenly distributed on the iRBC surface, and (B). PfEMP1 protein distribution was spotty (scale bar = µm). 48 Optical and fluorescence microscopic images of Alexa Fluor 488 stained CSA on CHO cells. Without CSA antibody, Alexa Fluor 488 stained cannot be observed as shown on the left panels, suggesting the fluorescent staining is specific to CSA (scale bar = 10µm). 49 Adhesion force measured at contact durations of 5s, 30s and 50s. There is no significant change between three adhesion forces, suggesting that the adhesion had rapidly formed between two cells upon contact. 50 Optical microscope images of (A) nRBC, (B) Ring stage iRBC (red arrow indicates a ring shaped structure), (C) Early trophozoite stage iRBC (the PV, as indicated by the red arrow, radius is less than one third of the total cell radius), (D) Late trophozoite stage iRBC (the PV radius reaches 50% of the total cell radius), (E) Schizont stage iRBC (the PV radius is equal or larger than 50% of the total cell radius and a clear crystallized hemozoin was visible under microscope), and (F) Late schizont stage iRBC (the PV almost occupies the whole cell). 52 viii Figure 2.13. Figure 2.14. Figure 2.15. Figure 2.16. Figure 2.17. Figure 2.18. Figure 2.19. Figure 2.20. Figure 2.21. Figure 2.22. Percentage of adhesion between nRBCs, and iRBCs at ring, early trophozoite, late trophozoite, early schizont and late schizont stages to CSA-expressing CHO cells. No adhesion was obtained between nRBCs to CHO cells, and ring stage iRBCs to CHO cells. The experiments were carried out at room temperature with binding target of CSA on CHO cells (Tests were carried out on diffierent days for each group). 54 Adhesion forces of early trophozoite, late trophozoite, early schizont and late schizont stages iRBCs to CHO cells. While early schizont stage iRBCs exhibited a significantly higher adhesion force (p[...]... 1.3 The biconcave shape of normal red blood cells observed under the optical microscope (scale bar = 5 µm) 7 The interior of the RBCs comprises hemoglobin, and the cell deformability and durability is determined by its unique structure of the cell membrane (Fung et al 1968; Evans 1973) Thus, the material properties of the red blood cell membrane are of interest in studying its deformability The red blood. .. crosslink with the red blood cell spectrin network, and reduce the RBC deformability In order to avoid spleen clearance of the rigid infected RBCs (iRBCs), another parasite exported protein - PfEMP1, is exported to the iRBC membrane The internal domain of PfEMP1 binds to KAHRP and PfEMP3, and the external binding domains are extruded out of the knob structure It acts as the adhesion ligand of iRBCs, and... normal red blood cells iRBCs infected red blood cells KAHRP knob associated histidine rich protein PAM pregnancy-associated malaria PfEMP1 Plasmodium falciparum RBC membrane protein 1 PfEMP3 Plasmodium falciparum RBC membrane protein 3 PS phosphatidylserine PV parasitophorous vacuole RBCs red blood cells RESA ring -infected RBC surface antigen RSP-2 ring surface protein-2 xiii List of Symbols ΔLp the aspirated... aspiration ΔP the applied pressure in micropipette aspiration D the cell-cell contact diameter g gravitational acceleration F the force exerted by the micropipette acting on the cell h the height of the water column L the change of the voltmeter reading corresponding to 2 cm change in the water column height in the dual pipette force measurement p the water pressure r the micropipette inner radius Rp the micropipette... parasites, such as Plasmodium falciparum RBC membrane protein 3 3 (PfEMP3), PfEMP1 and knob associated histidine rich protein (KAHRP), modify the host RBCs deformability and adherent properties The asexual stage is considered to be important to parasite multiplication inside the human body, and the altered mechanical properties of infected red blood cells (iRBCs) are considered to be the main cause of malaria... slide between each other during cell deformation Thus, the lipid bilayer contributes little to the resistance of the cell to deformation The inner spectrin network consists of the structural proteins, and it connects and supports the lipid bilayer It also defines the shape of the red blood cell, and determines the cell membrane shear elastic modulus (Evans 1973; Evans et al 1976) The spectrin network... considered as a continuum in the plane of the membrane, and on which the "stress resultants" or in-plane "tension" was defined The surface properties represented a summation of the properties of lamellar molecular structures over the thickness of the membrane With this model and micropipette aspiration technique, the shear elastic modulus of nRBCs at room temperature was measured to be 6 to 9 pN/µm, and the. .. used in micropipette aspiration Tm the cell membrane tension V the voltage reading on voltmeter wa the adhesion energy density ρ the density of water µ the membrane elastic shear modulus π the ratio of a circle's circumference to its diameter θa the angle formed between the stretched cell membrane and the normal of the CHO adhesive substrate θm the angle formed between the stretched cell membrane and... understand malaria -infected RBCs cytoadherent property, and to produce new drugs aiming at the anti-adherence of infected RBC 1.3 Current Studies on the Mechanical Properties and Cytoadherence of Infected RBCs 1.3.1 Mechanical Properties of Malarial -Infected RBCs a Healthy RBCs The red blood cell (RBC) is an essential biological cell that acts as an oxygen carrier to the different organs in the human body... mosquito-borne infectious disease that is caused by the eukaryotic of the genus Plasmodium There are five species of human Plasmodium, namely, Plasmodium falciparum (P falciparum) , P ovale, P vivax P malariae and P knowlesi, Among the five species, P falciparum is the most deadly causing more than 90% of malaria induced death (Despommier et al 2000) People suffered from malarial fever as far back as 1500 B.C . INVESTIGATING THE CYTOADHERENCE OF PLASMODIUM FALCIPARUM INFECTED RED BLOOD CELLS ZHANG ROU (B.Eng. (Hons), NTU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. and bind to the endothelial cells which results in vascular occlusion. The cytoadherence of malaria -infected red blood cells (iRBCs) is directly related to the malaria severity, and the occlusion. by the eukaryotic of the genus Plasmodium. There are five species of human Plasmodium, namely, Plasmodium falciparum (P. falciparum) , P. ovale, P. vivax P. malariae and P. knowlesi,. Among the